Functional Characterization of Aquaporin-4 Specific T Cells: Towards a Model for Neuromyelitis Optica

Antibodies to the water channel protein aquaporin-4 (AQP4), which is expressed in astrocytic endfeet at the blood brain barrier, have been identified in the serum of Neuromyelitis optica (NMO) patients and are believed to induce damage to astrocytes. However, AQP4 specific T helper cell responses that are required for the generation of anti-AQP4 antibodies and most likely also for the formation of intraparenchymal CNS lesions have not been characterized. specific T cells were present in the natural T cell repertoire of wild type C57BL/6 mice and T cell lines were raised. However, active immunization with these AQP4 peptides did not induce signs of spinal cord disease. Rather, sensitization with AQP4 peptides resulted in production of IFN-γ, but also IL-5 and IL-10 by antigen-specific T cells. Consistent with this cytokine profile, the AQP4 specific antibody response upon immunization with full length AQP4 included IgG1 and IgG2, which are associated with a mixed Th2/Th1 T cell response. restricted AQP4 specific T cell epitopes will allow us to investigate how AQP4 specific autoimmune reactions are regulated and to establish faithful mouse models of NMO that include both cellular and humoral responses against AQP4

In vivo imaging reveals rapid astrocyte depletion and axon damage in a model of neuromyelitis optica-related pathology.

OBJECTIVE: Neuromyelitis optica (NMO) is an autoimmune disease of the CNS, which resembles multiple sclerosis (MS). NMO differs from MS, however, in the distribution and histology of neuroinflammatory lesions and shows a more aggressive clinical course. Moreover, the majority of NMO patients carry IgG autoantibodies against aquaporin-4 (AQP4), an astrocytic water channel. Antibodies against AQP4 can damage astrocytes via complement, but NMO histopathology also shows demyelination, and - importantly - axon injury, which may determine permanent deficits following NMO relapses. The dynamics of astrocyte injury in NMO and the mechanisms by which toxicity spreads to axons are not understood. METHODS: Here, we establish in vivo imaging of the spinal cord, one of the main sites of NMO pathology, as a powerful tool to study the formation of experimental NMO-related lesions caused by human AQP4 antibodies in mice. RESULTS: We found that human AQP4 antibodies caused acute astrocyte depletion with initial oligodendrocyte survival. Within two hours of antibody application, we observed secondary axon injury in the form of progressive swellings. Astrocyte toxicity and axon damage were dependent on AQP4 antibody concentration and complement, specifically C1q. INTERPRETATION: In vivo imaging of the spinal cord reveals the swift development of NMO-related acute axon injury following AQP4 antibody-mediated astrocyte depletion. This approach will be useful in studying the mechanisms underlying the spread of NMO pathology beyond astrocytes, as well as in evaluating potential neuroprotective interventions. This article is protected by copyright. All rights reserved.peerReviewe

Peptide pools.

<p>Peptide pools.</p

Immunodominant I-A^b restricted epitopes of AQP4 and sequence homology of human vs mouse AQP4.

<p>(A) Topological model of AQP4 (M1 translational isoform) according to Crane and coworkers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016083#pone.0016083-Crane1" target="_blank">[8]</a>. The core immunogenic (I-A^b restricted) determinant of AQP4 in the N-terminus is highlighted in dark blue. The remainder of the immunogenic T cell epitopes is highlighted in cyan. The numbers indicate the amino acid residue position in the AQP4 protein sequence. (B) Amino acid sequence alignment of human and mouse AQP4 M1 isoforms. I-A^b restricted T cell determinants are highlighted (cyan). The dominant immunogenic epitope is represented by peptide 8 as indicated by a box (peptide sequence in dark blue). T cell epitopes common to M1 and M23 isoforms of AQP4 are underlined. Asterisks indicate sequence identity at the corresponding sequence position.</p

Overlapping AQP4 peptides.

<p>Overlapping AQP4 peptides.</p

Screening of T cell responses with pools of AQP4 peptides.

<p>Mice were immunized with full length AQP4 protein/CFA. Draining lymph node cells and splenocytes were restimulated <i>in vitro</i> with pools of overlapping AQP4 peptides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016083#pone-0016083-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016083#pone-0016083-t002" target="_blank">2</a>). (A) Proliferative responses of AQP4-sensitized lymphocytes to 21 pools of overlapping AQP4 peptides as measured by ³[H] thymidine incorporation (c.p.m.). Mean of triplicate cultures + SD or stimulation indices as calculated by deviding the c.p.m. values of each peptide pool by the background c.p.m. This experiment was performed twice with similar results. (B) In a second round of restimulation, T cells out of each pool were stimulated with the individual peptides of the parental pool in the presence of irradiated syngeneic splenocytes as APCs. Proliferative responses of T cell pools that showed antigen specific responses to at least one individual splenocytes with a stimulation index of at least 3.0 are depicted. Mean of ³[H] thymidine incorporation (c.p.m.) of triplicate cultures + SD are shown.</p

Functional characterization of AQP4_22–36 peptide specific TCL8.2.

<p>Representative T cell line (TCL) reactive to AQP4_22–36. (A) TCL8.2 cells were restimulated with irradiated syngeneic splenocytes in the presence of increasing concentrations of AQP4_22–36. The proliferative response was determined by ³[H] thymidine incorporation. Mean c.p.m. of triplicate cultures + SD. (B) Cytokine production of TCL8.2 cells in response to increasing concentrations of AQP4_22–36 as measured by cytometric bead array in the cell culture supernatant collected at 48 h after initiation of restimulation. Mean cytokine concentrations + SD of triplicate cultures. (C) Intracellular cytokine staining of TCL8.2 after 6 restimulation cycles.</p

Antibody response to AQP4.

<p>Wild type C57BL/6 mice were immunized with full length AQP4/CFA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016083#s4" target="_blank">Materials and Methods</a>. Unimmunized mice or mice immunized with MOG_35–55/CFA were used as controls. Sera of mice from each group were collected and tested for AQP4 specific antibodies in a cell based flow cytometry analysis. Subclass specification was performed by using fluorochrome labeled anti-mouse Ig antibodies specific for IgM, IgA, IgE, IgG1, IgG2a, IgG2b, and IgG3 (FITC labeled) or total IgG H+L (AlexaFluor488 labeled). (A) Representative histogram plots illustrating the MFIs for AQP4 specific IgG H+L in the various test groups. (B) Representative histogram plots of AQP4 specific Ig classes and IgG subclasses in AQP4 immunized animals. (C) ΔMFIs + SEM (n = 4) for AQP4 specific IgG H+L in naive control mice, MOG_35–55 immunized mice, or full length AQP4 immunized mice. (D) ΔMFIs + SEM (n = 4) for individual anti-AQP4 Ig classes and IgG subclasses in full length AQP4 immunized mice.</p